1. Field of the Invention
This invention relates generally to the field of automated sample processing, and more particularly to the isolation of rare cells from body fluids by an automated device for further analysis as it relates to the diagnosing, monitoring and managing of specific diseases, particularly cancer.
2. Background Art
The principal methodology and apparatus of the present invention stems from prior art assessing the detection and enumeration of cells in biological samples. These biological samples include, for example, blood, lymphatic fluid, or cerebral spinal fluid. A biological sample can also include buffer with spiked cultured cells, used as a control.
Manual methods for the isolation and quantification of rare cells of in peripheral blood and other biological matrices have been published and are well known in the art. However, to create sensitive, reproducible and high throughput testing protocols suitable for clinical diagnosis, an automated process is necessary.
It is well known that, for highly complex laboratory procedures, the root cause of erroneous results can frequently be traced to the cumulative effects of systematic or random pre-analytical errors, i.e. errors occurring during sample preparation or pre-processing stages rather than to the analytical method itself. Pre-analytical errors may manifest as variations due to technique-sensitive process steps as well as normal random or systematic variations from operator to operator. Sample preparation, specifically cell enrichment, is an example of a pre-analytical process which, when performed inconsistently, will manifest itself in high variability of assay results. Hence, automating such pre-analytical steps would minimize variability and result in more consistent analytical results.
Cell enrichment techniques for the isolation of rare cells vary widely and involve a variety of manual techniques to selectively isolate target cells for analysis. Centrifugation, with or without density gradients, is a common method employed. However, centrifugation does not easily lend itself to automation without the addition of complex robotic handling of sample tubes, computer-controlled centrifuges, etc. Often times, manual wash steps also involving centrifugation steps follow these procedures. If an approach were taken to automate all of these manual procedures directly, the end result would be a highly complex and expensive system that would mimic the manual processes performed by a skilled operator.
Magnetic separation of immuno-magnetically labeled target entities directly from the specimen is the ideal mode, but frequently inefficient if the target entity is a minor constituent in a complex mixture. Pre-purification by centrifugation or gradient separations followed by magnetic labeling and collection may be necessary in such cases. Again, these approaches do not lend themselves to facile automation without the addition of complex robotic handling systems for sample tubes, computer-controlled centrifuges, etc. Manual wash steps also involving centrifugation steps often follow these procedures. Again, direct automation of these manual procedures would result in a highly complex and expensive robotic system mimicking a manual process where every process step increases the risk of cell loss.
Several companies offer semi-automated systems specifically adapted to cell sorting or selection with application to cancer diagnostics and treatment. Miltenyi Biotech, Germany, has developed the autoMACS system, a semi-automated bench top magnetic cell sorter for collecting magnetically labeled target cells that are subsequently further sorted or fractionated on a flow cytometer into various cell populations including presumed target cells. However, the AutoMACS requires an enriched fraction prepared by manual pre-purification of complex specimens, e.g. blood, to remove most of the non-target cells. Flow cytometry also detects fluor-labeled particulate events of certain sizes not cells, i.e. without morphological confirmation of the identity of the detected events as target cells. U.S. Pat. No. 6,046,585, issued to Quantum Design, San Diego, discloses methods and apparatus for sensing and measuring small quantities of magnetic particles bound to target entities including cells; however, these entities are not considered rare.
The semi-automated method and apparatus, previously described (U.S. patent application Ser. No 10/081,996), is primarily designed to enrich circulating cells from blood and other specimens with a greater precision than attainable with the aforementioned manual methods. This is accomplished primarily by replacing imprecise manual steps such as vortex/mixing and quantitative fluid transfers with magnetic enrichment and magnetic washing, thus removing the steps typically involved in centrifugal density gradient separation. Aspiration steps normally performed manually were automated as well. The procedure required operator intervention periodically throughout the process, thus leading to variable process timing. While the system has general utility in sample processing of diverse materials, particularly biological specimens in a research setting, a more automated system incorporating a system of checks to prevent reagent or sample loss would greatly improve preparation and increase the integrity of the sample for clinical use.
The disclosed apparatus is a substantial improvement to the semi-automated detection device of Ser. No. 10/081,996. In the present application, the operator interaction during processing is eliminated, thus reducing associated errors. Sample preparation time is reduced and standardized with more precise control of individual processing steps. The improved system provides an enriched fraction suitable for detection, enumeration and identification of target cells by various analytical methodologies. The presence and quantities of such target cells in a sample specimen can be utilized for screening and detection in multiple types of diseases such as, but not limited to, metastatic cancer and cardiovascular disorders. Further, the isolation and analysis of these target cells has diagnostic importance in assessing multiple aspects of a disease, such as for example in cancer with early stage pre-metastatic cancer, monitoring for response to therapy and selection of more effective dose regimens or alternative therapies.